MICROSCOPY & IMAGE ANALYSIS OF UNSTAINED MACROMOLECULES

病毒是已知和研究最多的病原体之一。它们几乎感染了从细菌到人类的每一个活着的有机体，是密集的科学研究的对象。由于病毒是宿主的寄生虫，任何病毒的生命周期都与宿主细胞的生命周期密不可分。尽管存在这种依赖性，但所有病毒都有一些生存所必须完成的基本任务。病毒必须找到并识别能够在其中复制的细胞，将其基因组释放到细胞中，产生新的病毒组件，并将这些组件组装成前体，这些前体成熟为稳定的子代病毒粒子，从宿主细胞释放出来，然后传播以遇到新的宿主。病毒通过适应不同的细胞环境，以不同的方式完成这些任务。每一项任务都涉及整个病毒粒子范围内各组分之间的相互作用，因此需要对整个结构进行可视化，而低温透射电子显微镜(CryoTEM)和三维(3D)图像重建(“低温重建”)技术在这方面表现出色。我们将利用这些技术来研究各种各样的病毒，包括那些感染哺乳动物、昆虫、细菌和植物(包括藻类)的病毒。由目前的赠款资助的几项研究说明了不同病毒对病毒生命周期常见任务的结构性反应。这项建议包括继续和新的研究，重点是病毒和病毒复合体的结构调查以及超出当前结晶学技术领域的动态事件。我们的大量和广泛的研究是通过几个卓有成效的合作成为可能的，这些合作提供了来自生化、遗传学、免疫学和X射线结晶学实验的重要相关信息。我们的分析经常将来自3D CryoEM数据的信息与现有的原子模型结合起来。将原子模型与CryoEM 3D数据进行拟合，可以揭示抗体和受体分子在病毒衣壳表面的定向和结合位置的伪原子分辨率信息，这些信息可以通过分子遗传学实验进行测试和改进。将研究二十面体病毒和非二十面体病毒。这些病毒包括几个不同的病毒家族的代表，所有这些病毒都是研究形式和功能的优秀模式系统：呼肠孤病毒科、细小病毒科、短小病毒科、足状病毒科、双生病毒科、细小病毒科和虹彩病毒科。

Viruses are among the best known and studied pathogens. They infect virtually every living organism from bacteria to man and are the subjects of intensive scientific investigations. As viruses are parasites of their hosts, the life cycle of any virus is inextricably tied to that of the host cell. Despite this dependence, all viruses share a number of essential tasks which they must accomplish for survival. A virus must find and recognize a cell in which it can replicate, release its genome into the cell, generate new viral components and assemble these components into precursors that mature into a stable progeny virion which is released from the host cell and transmitted to encounter a new host. Viruses accomplish these tasks in different ways as a result of adaptation to different cellular environments. Each task involves interactions between components within the context of the whole virion and hence requires the visualization of the entire structure at which the techniques of cryo- transmission electron microscopy (cryoTEM) and three-dimensional (3D) image reconstruction ('cryo-reconstruction') excel. We will exploit these techniques to study a diverse range of viruses, including those that infect mammals, insects, bacteria, and plants (including algae). Several studies funded by the current grant have illustrated the structural response of different viruses to the common tasks of the viral life cycle. This proposal involves continued as well as new studies that focus on structural investigations of viruses and virus complexes and dynamic events that lie beyond the current realm of crystallographic technology. The large number and extent of our studies are made possible through several fruitful collaborations which provide important correlative information from biochemical, genetic, immunological, and X-ray crystallographic experiments. Our analyses often combine information from 3D cryoEM data with available atomic models. The fitting of atomic models to cryoEM 3D data can reveal 'pseudo-atomic' resolution information about the orientation and binding sites of antibody and receptor molecules on viral capsid surfaces which can be tested and refined by molecular genetics experiments. Icosahedral as well as non-icosahedral viruses will be studied. These include representatives of several different virus families, all of which serve as excellent model systems for studying form and function: Reoviridae, Parvoviridae, Picornaviridae, Podoviridae, Geminiviridae, Phycodnaviridae, and Iridoviridae.